The overall goal of these studies is to understand the endothelial nitric oxide synthase (eNOS) signaling system in health and disease. Regulation of endothelial NO production is a key determinant of blood pressure, platelet aggregation, and angiogenesis. The fundamental hypothesis of this research program is that eNOS post-translational modifications and protein-protein associations are essential determinants of NO-dependent signaling in the vascular wall. During the previous funding period, we identified plasmalemmal caveolae as the subcellular organelle to which eNOS is reversibly targeted; characterized the enzyme responsible for depalmitoylation of eNOS; discovered a receptor-modulated regulatory cycle for eNOS involving the enzyme's reversible interactions with caveolin and calmodulin; identified a new receptor pathway for eNOS activation involving the platelet-derived sphingolipid sphingosine I-phosphate (S1P) ; and explored the roles of MAP kinases and phosphoinositide 3-kinase (P13-K)/kinase Akt in eNOS regulation. The proposed experiments will determine the relationships between MAP kinase and P13-Klkinase Akt pathways in eNOS regulation. Informative eNOS phosphorylation and palmitoylation mutants will be constructed, and features of their targeting and regulation will be studied. We plan to characterize the phosphoprotein phosphatase(s) that dephosphorylate eNOS. We will explore the regions of eNOS that modulate its interactions with caveolin, and will study the regulation of eNOS palmitoylation pathways by caveolin, NO, and acyl protein palmitoylthioesterase. We will explore the relationships between the newly-discovered eNOS agonist, S1P, and other receptor-modulated eNOS pathways, including the protein kinases that regulate NO signaling. We will explore the hypothesis that S1P-mediated angiogenesis is modulated by eNOS, and investigate the role of eNOS in mediating responses to angiogenesis inhibitors. Our studies of the cellular and molecular mechanisms that regulate eNOS may lead to the identification of new points for pharmacological intervention.